Without limiting the scope of the invention, its background is described in connection with oligonucleotide agents and with methods for the isolation of sequences that are bound by nucleic acid binding molecules and the like.
Virtually all organisms have nuclease enzymes that degrade rapidly foreign DNA as an important in vivo defense mechanism. The use, therefore, of normal oligonucleotides as diagnostic or therapeutic agents in the presence of most bodily fluids or tissue samples is generally precluded. It has been shown, however, that phosphoromonothioate or phosphorodithioate modifications of the DNA backbone in oligonucleotides can impart both nuclease resistance and enhance the affinity for target molecules, such as for example the transcriptional regulating protein NF-κB. Thus, from the foregoing, it is apparent there is a need in the art for methods for generating aptamers that have enhanced binding affinity for a target molecule, as well as retained specificity. Also needed are ways to identify and quantify in detail the mechanisms by which aptamers interact with target molecules.
Current DNA array technology is problematic in that it is focused on the identification and quantification of a single mRNA species, and does not provide information on the more relevant level of functional protein expression and in particular protein-protein interactions such as between heterodimers and homodimers. Although microarrays have been used for detecting the proteome, most of these are based on antibodies or normal backbone aptamers.
Synthetic phosphodiester-modified oligonucleotides such as phosphorothioate oligonucleotide (S—ODN) and phosphorodithioate oligonucleotide (S2—ODN) analogues have increased nuclease resistance and may bind to proteins with enhanced affinity. Unfortunately, ODNs possessing high fractions of phosphorothioate or phosphorodithioate linkages may lose some of their specificity and are “stickier” towards proteins in general than normal phosphate esters, an effect often attributed to non-specific interactions. The recognition of nucleic acid sequences by proteins involves specific sidechain and backbone interactions with both the nucleic acid bases as well as the phosphate ester backbone, effects which may be disrupted by the non-specific interactions caused with S—ODN and S2—ODN analogues.
Gorenstein, et al., U.S. Pat. No. 6,423,493, have taken advantage of this “stickiness” to enhance the affinity of S—ODN and S2—ODN agents for a protein target. A screening method was developed to optimize the total number of phosphorothioate or phosphorodithioate linkages that: decreased non-specific binding to the protein target while enhancing only the specific favorable interactions with the target protein.
Other advances in combinatorial chemistry allow construction and screening of large random sequence nucleic acid “aptamer” libraries (e.g., Ellington, A. D. and Szostak, J. W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature, 346, 818-822); targeting proteins (e.g., Bock, L. C., et al., (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature, 355, 564-566.); and other molecules (Koizumi, M. and Breaker, R. R. (2000) Molecular recognition of cAMP by an RNA aptamer. Biochemistry, 39, 8983-8992; Gold, L., et al. (1997) SELEX and the evolution of genomes. Curr. Opin. Genetic. Dev., 7, 848-851.; and Ye, X., et al. (1996) Deep penetration of an α-helix into the widened RNA major groove in the HIV-1 Rev peptide-RNA aptamer complex. Nat. Struct. Biol., 3, 1026-1033.).
The identification of specific S—ODN and S2—ODN aptamers (“thioaptamers”) that bind proteins based upon in vitro combinatorial selection methods, however, is limited to substrates only accepted by polymerases required for reamplification of selected libraries by the polymerase chain reaction (PCR). One disadvantage of using the polymerization of substituted nucleoside 5′-triphosphates into ODN aptamers are the restrictions on the choice of P-chirality by the enzymatic stereospecificity. For example, it is known that [Sp]-diastereoisomers of dNTP(αS) in Taq-catalyzed polymerization solely yield [Rp]-phosphorothioate stereoisomers (Eckstein, F. (1985) Nucleoside phosphorothioates. Annu. Rev. Biochem., 54, 367-402.). Therefore, using current methods it is not possible to select [Sp]-phosphorothioate stereoisomers along with achiral S2—ODN analogous since both [Rp]-diastereoisomers of dNTP(αS) and nucleoside dNTP(αS2) are not substrates of polymerases. Additionally, these in vitro combinatorial selection methods require many iterative cycles of selection and reamplification of the bound remaining members of the library by the PCR, which are quite time consuming.
What is needed are compositions and methods that permit the isolation of, e.g., individual aptamer:protein complexes without the need for repeated iterative cycles of selection and reamplification of likely binding targets. Also needed are compositions, methods and systems that permit the creation, isolation, sequencing and characterization of making [Sp]-phosphorothioate stereoisomers along with achiral S2—ODN analogs using, e.g., [Rp]-diastereoisomers of dNTP(αS) and nucleoside dNTP(αS2). Also needed are methods for creating libraries that permit not only the isolation of a primary aptamer:protein target, but the isolation of protein(s) that may interact with the aptamer:protein target, so called secondary interactions.